Regulatory T (TR) cells that express the transcription factor Foxp3 play a requisite role in peripheral immunological tolerance and in restraining exuberant immune responses. Elucidating mechanisms that control TR cell responses is of pivotal importance to the restoration of tolerance in diseases of chronic inflammation and autoimmunity. Relevant to this goal is our recent determination of a critical role for the Notch pathway in regulating the peripheral TR cell compartment. TR cell-specific constitutive expression of the intracellular domain of Notch1 (Notch1c) led to lymphoproliferation Th1 cytokine dysregulation and autoimmunity. These changes were reflected in alterations in the TR cell transcriptome, with reduced expression of several key TR cell markers, and decreased TR cell suppressive functions both in vitro and in vivo. In contrast, TR-specific loss of function mutations that impair Notch signaling, including the deletion of Pofut1, Rbpj and Notch1, led to the contraction of the peripheral T cell compartment and suppression of Th1 response. Some of the effects of Notch signaling in TR cells involved transcriptional activation via the canonical Notch pathway co-factor RBPJ. Yet others appeared to involve activation of the non-canonical mTORC2/AKT/Foxo1 pathway, evidenced by enhanced phosphorylation of the mTORC2 target AKT S473 and augmented translocation of Foxo1 to the cytosol upon TCR/CD3 activation. Accordingly, we hypothesize that Notch signaling fulfills an important physiological function in reversibly regulating TR cell responses to ensure optimal immune homeostasis. Excessive activation of this pathway would result in TR cell dysfunction, inflammation and autoimmunity. Accordingly, we propose to establish the physiological functions of Notch signaling in TR cells, including its regulation of TR cell activity in primary immune responses, its role in conferring TR cell fitness, and the identity of the interacting ligands. We also propose to determine the contribution of canonical and non- canonical transcriptional mechanisms to TR cell regulation by Notch signaling. Finally, we will elucidate the consequences of Notch1c-mediated Th1 reprogramming of TR cells on their control of Th1 responses, and the role of T follicular regulatory (TFR) cell subversion by Notch1c is fostering dysregulated humoral autoimmunity. The proposed studies will identify novel mechanisms involving the Notch pathway that control the TR cell response and the consequence of their disruption in fostering autoimmunity.